Antibiotic compounds

ABSTRACT

The present invention provides methods for identifying (3-amino-2-oxo-azetidin-1-yl) acetic acid derivatives with anti-PBP2a activity. The method involves the selection of RNA biocatalysts that promote the formation of (3-amino-2-oxo-azetidin-1-yl) acetic acid derivatives with anti-PBP2a activity from component reactants. The invention also provides novel (3-amino-2-oxo-azetidin-1-yl) acetic acid derivatives with anti-PBP2a activity identified by these methods. The invention also provides RNA biocatalysts that are capable of catalyzing the formation of (3-amino-2-oxo-azetidin-1-yl) acetic acid derivatives from component reactants.

FIELD OF THE INVENTION

The present invention relates to antibiotic compounds, especially thoseeffective against methicilin-resistant Staphylococcus aureus, andmethods for their preparation.

BACKGROUND OF THE INVENTION

Methicillin-resistant Staphylococcus aureus (MRSA) is a prevalent andrapidly growing nosocomial pathogen problem. Not only has the incidenceof MRSA among hospital S. aureus isolates reached 50% (Am. J. Infect.Control 27: 520–532), there is also an emerging prevalence of MRSAstrains in the community (Chambers, et al., Emerging Infectious Diseases7: 178–182). The resistance of MRSA to methicillin and other β-lactamantibiotics is mediated by the acquired penicillin-binding protein 2a(PBP2a). PBP2a is a transpeptidase involved in cell wall peptidoglycanbiosynthesis and has very low affinity for these antibiotics. A rationalapproach to anti-MRSA drug development is to restore sensitivity toR-lactam antibiotics by directly targeting this molecular mechanism ofresistance.

Evolutionary Chemistry™ is a methodology for the discovery of smallmolecule pharmaceutical lead compounds. Evolutionary Chemistry™ isunique in that it integrates the steps of small molecule synthesis andhigh throughput screening into a single system. This is accomplished byutilizing the ability of RNA to catalyze chemical transformations thatcan create drug-like molecules, and exploiting this ability to assemblean enormous small molecule library. By incubating a large library ofreactant-coupled, random-sequence, modified RNAs (approximately 10¹⁵unique potential biocatalysts) with a library of small moleculereactants (10⁴–10⁶ unique constituents), a library of 10⁵–10⁸ potentiallead compounds can be generated. Potential lead compounds remainattached to the biocatalysts responsible for their formation and arethus addressable. The biocatalyst-assembled product library is thensubjected to evolutionary pressures that demand that the selected smallmolecules have specified properties (such as high affinity for a drugtarget). Biocatalysts conjugated to lead compounds that exhibit thedemanded properties are enzymatically amplified. The biocatalystsequence-specific small molecule assembly is reliably reproduced insubsequent cycles of biocatalysis, selection, and amplification. Thesecycles are iterated with increasing evolutionary pressure until the mosteffective lead compounds evolve from the population.

In the most general embodiments, a nucleic acid-reactant test mixture isformed by attaching a first reactant to each of the nucleic acids in atest mixture (containing 10² to 10¹⁸ nucleic acids with randomizedsequences). The nucleic acid-reactant test mixture is treated with otherfree reactants that will combine with the first reactant to formdifferent products. It is important to note that from the nucleic acidtest mixture, discrete nucleic acid sequences will be associated withfacilitating the formation of the different shaped products. Theproducts may differ in shape, reactivity or both shape and reactivity.Partitioning of the desirable product shape or reactivity isaccomplished by binding to or reaction with a target. Proteins, smallmolecules, lipids, saccarides, etc., are all examples of targets. Afterbinding to or reacting with the target the non-interacting products, arepartitioned from the interacting products, and discarded. The nucleicacid associated with the interacting product is then amplified by avariety of methods known to those experienced in the art. This nucleicacid is then used to facilitate the assembly of the desirable product byfacilitating the specific reaction to form the selected product ontreatment with the mixture of starting reactants. In a typical reaction,the amplified nucleic acid can be reattached to the first reactant,however, said reattachment is not always required. This is an idealizedcase and in many examples the nucleic acid facilitator may assemble morethan one product from the starting mixture, but all of the productsselected will have the desired properties of binding to or chemicalreaction with the target.

The overall process is described in more detail in, for example, U.S.Pat. Nos. 6,048,698; 6,030,776; 5,858,660;5,789,160; 4 5,723,592; and5,723,289, each of which is entitled “Parallel SELEX,” and each of whichis incorporated herein by reference in its entirety. These patents arehereinafter referred to collectively as the “Parallel SELEX patents.”

The present invention provides novel monobactams with anti-PBP2aactivity that were identified using the aforementioned EvolutionaryChemistry™ process.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides monobactams with anti-PBP2aactivity having the following formula:

wherein X is CH₂, NH, or O;

-   R₁, R₂, R₃, R₄ and R₅ are independently selected from the group    consisting of H, C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, OR⁶,    C(O)R⁶, carboalkoxyalkyl, heterocyclyl, aromatic hydrocarbon and    cycloalkyl, all of which may be optionally substituted by one or    more of the groups selected from C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl,    C₁–C₂₀ alkynyl, cycloalkyl, heterocyclyl, aryl, halogen, cyano,    nitro, amino, alkylamino, dialkylamino, aminoalkyl,    dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl,    alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁶, OR⁶, CONR⁶,    wherein all said substituents may be optionally substituted with one    or more selected from the group consisting of halogen, C₁–C₂₀ alkyl,    C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, cycloalkyl, OR⁶, C(O)R⁶,    carboalkoxyalkyl, cyano, and nitro; and-   R₆ is selected from the group consisting of hydrogen, halogen,    C₁–C₂₀ alkyl, aromatic hydrocarbon, and alkylaryl, wherein all said    substituents may be optionally substituted by one or more    carboalkoxy, thiol, amino, hydroxyl, carboxyl, lower alkyl, lower    alkenyl, lower alkynyl, halo, cyano, nitro, carboxyalkyl, and    carboxamides.

The invention also provides methods for identifying monobactams withanti-PBP2a activity. The methods employ RNA biocatalyst libraries inwhich each RNA library member has a randomized sequence region and aunique sequence region that encodes the identity of a tethered dienereactant. The RNA molecules are incubated with free reactants formed bythe cyclotrimerization of a monobactam alkyne with additional alkynes,at least some of which additional alkynes bear a dienophilefunctionality. RNA molecules that catalyze the Diels-Alder reactionbetween a diene and a cyclotrimerization product with a dienophilefunctionality yield a product that binds to PBP2a (which product istethered to the 5′ end of the RNA via the diene) and are partitionedfrom the library by virtue of the affinity of the tethered product forPBP2a. The RNA molecules are then amplified, and used to initiatefurther cycles of selection, leading to the identification of 1) amonobactam that binds to PBP2a; and 2) an RNA molecule (hereinafterreferred to as an “RNA biocatalyst”) that catalyzes the formation ofthat monobactam from a diene and a cyclotrimerization product (whichacts as a dienophile). The monobactam is then characterized bydeconvolution of the reaction history of the RNA, thereby yielding theidentity of the individual components incorporated into the monobactami.e., the alkynes used in the cyclotrimerization and the diene used inthe biocatalyzed Diels-Alder reaction.

The invention also provides RNA biocatalysts that can catalyze theformation of compositions with anti-PBP2a activity when tethered tospecific diene reactants and then incubated with the cyclotrimerizationproducts of specific alkynes. The compositions thereby produced are alsoincluded in the invention, as are the individual compounds within thecomposition that are responsible for the anti-PBP2a activity.

The invention also provides RNA biocatalysts that can catalyze theformation of compositions with anti-PBP2a activity in the absence oftethered diene reactants when incubated with the cyclotrimerizationproducts of specific alkynes. The compositions thereby produced are alsoincluded in the invention, as are the individual compounds within thecomposition that are responsible for the anti-PBP2a activity.

In another embodiment, the invention provides a monobactam compoundhaving the following formula:

wherein each n is independently 0–4; each X is independently O, S, CHZor NH; each R is independently lower alkyl optionally substituted withOR, where R₁ is H or lower alkyl; and each Z is independently H;halogen; OH; phenyl, heteroaromatic, or lower alkyl optionallysubstituted with one or more halogen, OH, phenyl or heteroaromaticgroups. The invention also includes methods for synthesizing thiscompound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically the selection process used to identifythe biocatalysts of the invention.

FIG. 2 provides sequence alignments for the random regions of individualclones from the 3H biocatalyst subpopulation.

FIG. 3 illustrates the PBP2a inhibitory activity of products produced byindividual biocatalysts isolated from subpopulation 3H.

FIG. 4 provides sequence alignments for the random regions of individualclones from the 8I biocatalyst subpopulation.

FIG. 5 illustrates the general structure of the RNAs used in thebiocatalyst screening method.

FIG. 6 illustrates the PBP2a inhibitory activity of products produced byparticular biocatalyst subpopulations and free reactant sublibraries.

FIG. 7 illustrates that the inhibition of PBP2a activity observed forproducts produced by the 3H biocatalyst subpopulation is not sensitiveto RNase digestion,

FIG. 8 illustrates a phylogenetic analysis of individual biocatalystsfrom the 3H subpopulation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The monobactams of the invention may be defined by the formula:

wherein X is CH₂, NH, or O;

-   R₁, R₂, R₃, R₄ and R₅ are independently selected from the group    consisting of H, C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, OR⁶,    C(O)R⁶, carboalkoxyalkyl, heterocyclyl, aromatic hydrocarbon and    cycloalkyl, all of which may be optionally substituted by one or    more of the groups selected from C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl,    C₁–C₂₀ alkynyl, cycloalkyl, heterocyclyl, aryl, halogen, cyano,    nitro, amino, alkylamino, dialkylamino, aminoalkyl,    dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl,    alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁶, OR⁶, CONR⁶,    wherein all said substituents may be optionally substituted with one    or more selected from the group consisting of halogen, C₁–C₂₀ alkyl,    C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, cycloalkyl, OR⁶, C(O)R⁶,    carboalkoxyalkyl, cyano, and nitro; and-   R₆ is selected from the group consisting of hydrogen, halogen,    C₁–C₂₀ alkyl, aromatic hydrocarbon, and alkylaryl, wherein all said    substituents may be optionally substituted by one or more    carboalkoxy, thiol, amino, hydroxyl, carboxyl, lower alkyl, lower    alkenyl, lower alkynyl, halo, cyano, nitro, carboxyalkyl, and    carboxamides.

The monobactams of the invention may also be defined as the reactionproducts formed by:

1) providing a monobactam core alkyne (also referred to herein as an “Aalkyne”) having the structure

wherein R₁ is one of:

R₂ is one of:

R₃ and R₄ are independently selected from the group consisting ofhydrogen, C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, OR⁵, C(O)R⁵,carboalkoxyalkyl, heterocyclyl, aromatic hydrocarbon and cycloalkyl, allof which may be optionally substituted by one or more of the groupsselected from C₁–C₂₀ alkyl, C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, cycloalkyl,heterocyclyl, aryl, halogen, cyano, nitro, amino, alkylamino,dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,alkylaminoaryl, alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁵, OR⁵,CONR⁵, wherein all said substituents may be optionally substituted withone or more selected from the group consisting of halogen, C₁–C₂₀ alkyl,C₁–C₂₀ alkenyl, C₁–C₂₀ alkynyl, cycloalkyl, OR⁵, C(O)R⁵,carboalkoxyalkyl, cyano, and nitro; and

-   R₅ is selected from the group consisting of hydrogen, halogen,    C₁–C₂₀ alkyl, aromatic hydrocarbon, and alkylaryl, wherein all said    substituents may be optionally substituted by one or more    carboalkoxy, thiol, amino, hydroxyl, carboxyl, lower alkyl, lower    alkenyl, lower alkynyl, halo, cyano, nitro, carboxyalkyl, and    carboxamides:-   2) reacting the monobactam alkyne of 1) with one or more of the    following alkynes (hereinafter referred to as “C alkynes”) under    conditions that promote alkyne cyclotrimerization:

with the priviso that when R₁ is one of:

the cyclotrimerization reaction mixture also includes one or more of thefollowing alkynes (hereinafter referred to as “B alkynes”):

-   3) performing a Diels-Alder reaction between the cyclotrimerization    product of 2) (a dienophile) and one of the following diene    reactants (dienes reactants 1–20):

The invention includes all monobactams with anti-PBP2a activity that maybe produced by the aforementioned cyclotrimerization and Diels-Alderreactions using the aforementioned reagents. The invention also includesmixtures comprised of a plurality of monobactams according to formula(b), wherein the individual monobactams in the mixture differ from eachother in at least one of the substituents R₁–R₄.

More specifically, the monobactams may be identified by preparing onemore more free reactant libraries of the aforementionedcyclotrimerization products, and then reacting each free reactantlibrary with one or more of the aforementioned diene reagents tetheredto the 5′ end of RNA molecules. The dienes may be tethered to RNA via apolethylene glycol (PEG) linker, the point of attachment of the PEGlinker to the dienes is shown below:

The RNA molecules form a RNA biocatalyst library in which each RNAlibrary member has a randomized sequence region and a unique sequenceregion that encodes the identity of the tethered diene. RNA moleculesthat catalyze the Diels-Alder reaction between a diene and acyclotrimerization product to yield a product that binds to PBP2a (whichproduct is tethered to the 5′ end of the RNA via the diene) arepartitioned from the library by virtue of the affinity of the tetheredproduct for PBP2a. The RNA molecules are then amplified, and used toinitiate further cycles of selection, leading to the identificationof 1) a monobactam that binds to PBP2a; and 2) an RNA molecule(hereinafter referred to as an “RNA biocatalyst”) that catalyzes theformation of that monobactam from a diene and a cyclotrimerizationproduct (which acts as a dienophile). The monobactam is thencharacterized by deconvolution of the reaction history of the RNA,thereby yielding the identity of the individual components incorporatedinto the monobactam i.e., the alkynes used in the cyclotrimerization andthe diene used in the biocatalyzed Diels-Alder reaction. Methods for theselection of RNA molecules that can generally catalyze the reaction of atethered reactant with a free reactant, and specifically can catalyzethe reaction between a tethered diene reactant and a free dienophilereactant, are provided in the Parallel SELEX patents. Examples 1–12below provide detailed and non-limiting descriptions of the methods usedto generate free reactant libraries, select RNA catalysts, and assay forPBP2a inhibition. FIG. 1 illustrates the selection processschematically.

More specifically, monobactams of the instant invention may berepresented by the formula:

wherein X is CH₂, NH, or O;

-   -   R₁ is the Diels-Alder product formed by the reaction of one of        diene reactants 1–20 (which may be tethered to a RNA        biocatalyst) with the functionality on the following B alkynes:

-   R₂ is the functionality found on the A alkynes, B alkynes or C    alkynes; and R₃ is one of:

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (c), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₆.

Note that the term “functionality” used in the context of A, B, or Calkynes refers to the moiety attached to the alkyne group, which moietybecomes one of the substituents of the ring formed duringcyclotrimerization. For example, in the following hyothetical thefunctionalities are Z₁, Z₂, and Z₃:

Additional monobactams of the instant invention may be represented bythe formula:

wherein R₁ is the Diels-Alder product formed by the reaction of one ofthe following diene reagents (shown as free amines; attachment to a RNAbiocatalyst is also contemplated, as discussed above):

with the functionality on one of the B alkynes, and wherein R₂ can bethe functionality found on an A, B, or C alkyne.

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (d), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₂.

Still further monobactams of the instant invention can be represented bythe formula:

wherein R₁ is the Diels-Alder product formed by the reaction of thefollowing diene reagent (shown as a free amine; attachment to a RNAbiocatalyst is also contemplated, as discussed above):

with the functionality on one of the B alkynes, and wherein R₂ can bethe functionality found on an A, B, or C alkyne.

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (e), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₂.

Even further monobactams of the instant invention can be represented bythe formula:

wherein R₁ is the Diels-Alder product formed by the reaction of thefollowing diene reagent (shown as a free amine; attachment to a RNAbiocatalyst is also contemplated, as discussed above):

with the functionality on one of the B alkynes, and wherein R₂ an be thefunctionality found on an A, B, or C alkyne.

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (f), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₂.

Yet further monobactams of the instant invention can be represented bythe formula:

wherein each of R₁ and R₂ is independently the functionality on any oneof the A alkynes, the B alkynes, and the C alkynes.

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (g), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₄.

The monobactams of the instant invention were initially identified byselecting for catalytic RNA molecules that catalyze the reaction betweena diene (tethered to the RNA via a PEG linker) and a dienophile (a freereactant library member). The selection process also identified RNAmolecules in which certain nucleophilic functionalities present in theRNA itself react with members of the free reactant library, therebyforming a monobactam that is tethered directly to the RNA, rather thanvia the diene tethered to a PEG linker. The following functionalitiespresent on RNA can serve as nucleophiles:

Hence, further monobactams of the instant invention can be representedby the formula

wherein R₁ is the product of the reaction between one of theaforementioned nucleophilic functionalties present in RNA with one ofthe functionalities on one of the B alkynes, and wherein R₂ an be anyfunctionality found on an A, B, or C alkyne.

The invention also includes mixtures comprised of a plurality ofmonobactams according to formula (h), wherein the individual monobactamsin the mixture differ from each other in at least one of thesubstituents R₁–R₂.

The invention also provides RNA molecules that can catalyze theformation of the above-mentioned compositions, and further includes thecompositions thereby produced. Preferred RNA molecules have thesequence:

5′ GGGAGAAUCAAAGUAAUCGCUCA-[X]- SEQ ID NO: 121 UUCGACAGGAGGCUCACAACAGGC3′in which X is one of the sequences provided in FIG. 2 (SEQ ID NOS: 41–84and 120), and U is 5-(4-pyridylmethyl) uridine). Each of theaforementioned RNA biocatalysts (hereinafter referred to collectively as“3H biocatalysts” may be used to provide a composition with anti-PBP2aactivity. Specifically, a free reactant library is synthesized bycyclotrimerizing, with all of the B alkynes and all of the C alkynes,the monobactam alkyne (A alkyne 43) having the formula:

An exemplary method for cyclotrimerization is provided in Example 2. Theresulting free reactant sublibrary (termed C43) comprises a large numberof cyclotrimerization products, each having the following formula:

wherein R₁ and R₂ are independently the functionality on a B alkyne, ona C alkyne, or:

Following cyclotrimerization, the free reactant sublibrary may bepartitioned from unreacted reagents. The library is then incubated withone or more of the aforementioned RNA biocatalysts. Biocatalysis yieldsa composition having anti-PBP2a activity. The individual biocatalyzedproducts in the composition with anti-PBP2a activity may then bepurified by virtue of their affinity for PBP2a. The structure of theproduct(s) responsible for the anti-PBP2a may be determined by, forexample, a combination of reactant library deconvolution, involving thesynthesis and analysis of successively smaller subsets of free reactantsublibrary C43, and tandem C18 reversed-phase HPLC-electrospray massspectrometry (LC-MS) techniques.

Preferably, the RNA biocatalyst used in conjunction with the freereactant library C43 is 3H4 (x=SEQ ID NO: 66), 3H15 (x=SEQ ID NO: 120),3H16 (x=SEQ ID NO: 77), 3H38 (x=SEQ ID NO: 75), 3H50 (x=SEQ ID NO: 62),3H56 (x=SEQ ID NO: 72), or 3H112 (x=SEQ ID NO: 43).

FIG. 3 illustrates percentage PBP2a inhibition values for thecompositions produced by these biocatalysts.

The reaction and kinetic parameters of PBP2a inhibition by thecompositions produced by these RNA biocatalysts with the free reactantlibrary C43 may be represented as follows:

${E + I}\mspace{14mu}\overset{k_{1}}{\underset{k_{- 1}}{\rightleftarrows}}\mspace{14mu}{{E \cdot I}\overset{k_{2}}{\rightarrow}{{E - I}\overset{k_{3}}{\rightarrow}{E \cdot I^{*}}}}$K₁ = k₃/(k₂/K_(m)); k₂ = (𝕕[E − I]/𝕕t)/[E ⋅ I]; K_(m) = [E][I]/[E ⋅ I]k₂/K_(m) = ((𝕕[E − I]/𝕕t)[E ⋅ I])/([E][I]/[E ⋅ I]) = (𝕕[E − I]/𝕕t)/([E][I])

Values for the individual kinetic parameters are listed in Table 1.

TABLE 1 Random Region [E] [I] [E − I] d[E − I] k₂/K_(m) K₁ IC₅₀ SEQ IDBiocatalyst (μM) (μM) (μM) (μM/min) (μM⁻¹min⁻¹) (μM) (μg/ml) NO: Entire3H 1.1 2.8 0.32 0.0036 0.0012 0.93 0.64 — subpop. (incl. tethered dienereactant 15) Clone 3H4 1.2 1.5 0.25 0.0028 0.0016 0.70 0.48 66 Clone3H15 1.2 1.5 0.34 0.0035 0.0021 0.53 0.37 120 Clone 3H16 1.5 2.0 0.340.0037 0.0012 0.93 0.64 77 Clone 3H38 1.2 1.5 0.38 0.0042 0.0023 0.480.33 75 Clone 3H50 1.2 1.5 0.19 0.0021 0.0011 0.97 0.67 62 Clone 3H561.2 1.5 0.27 0.0030 0.0017 0.68 0.47 72 Clone 3H112 1.5 1.5 0.33 0.00360.0016 0.69 0.48 43

The inhibition data indicates that the anti-PBP2a activity of theisolated monobactam derivatives produced by the aforementioned 3H4,3H15, 3H16, 3H38, 3H50, 3H56, and 3H112 RNA biocatalysts in conjunctionwith the free reactant sublibrary C43 is comparable to that observedwith bicyclic β-lactam inhibitors of this enzyme (literature IC₅₀ valuesrange from ˜0.4–4 μg/ml; examples given in Table 2; see Example 12).

TABLE 2 Examples of reported β-lactam inhibitors of PBP2a. Compound IC₅₀for Source Class PBP2a Reference Eli Lilly Carbacephem   2 μg/ml J. Med.Chem. 36: (LY-206763) 1971–76 Taiho 2-Thioisocephem 0.58 μg/mlChemotherapy 43: Pharmaceutical 1–5 (TOC-39) RW Johnson/ Cephem  0.5μg/ml ICAAC (1998) 38: Microside F14, 21, 22, 24, (MC-02479) &25

The individual biocatalyzed products in the composition with anti-PBP2amay then be purified by virtue of their affinity for PBP2a. Thestructure of the product(s) responsible for the anti-PBP2a may bedetermined by, for example, a combination of reactant librarydeconvolution, involving the synthesis and analysis of successivelysmaller subsets of free reactant sublibrary C43, and tandem C18reversed-phase HPLC-electrospray mass spectrometry (LC-MS) techniques.

Note that the anti-PBP2a activity generated by the individual RNAbiocatalyst subpopulation 3H clones with the free reactant sublibraryC43 does not depend on the presence of the tethered diene reactants 15and 16 (or of the associated PEG linker and 10 nt ssDNA). This indicatesthat the anti-PBP2a activity produced by these RNA biocatalysts is notthe result of a Diels-Alder reaction between a tethered diene and adienophile moeity on a free reactant sublibrary member. Instead, it islikely that a functional group inherent in RNA itself participates inthe biocatalyzed reaction with a free reactant sublibrary member,thereby acting as the “tethered” reagent that attaches the RNAbiocatalyst to the free reactant sublibrary member. As described above,the following groups in RNA can serve as tethered reagents:

More specifically, but without being limited to a single mechanism orhypothesis, it is contemplated that the primary amine group on an RNAbase reacts as a nucleophile with a functionality on a B or C alkyne.For example, the RNA biocatalyst may catalyze the following reactionbetween a functionality on a B alkyne (which functionality is present asa substituent of the phenyl ring of a free reactant sublibrary member)and an RNA base:

Example 10 and FIG. 7 demonstrate that the anti-PBP2a activity of thebiocatalyzed product is resistant to RNase digestion. Therefore, it islikely that only one or a very few RNA bases are required in the productfor activity.

In another embodiment, the invention provides further RNA molecules thatcan catalyze the formation of the above-mentioned compositions, andfurther includes the compositions thereby produced. Preferred RNAmolecules have the sequence:

5′dienel7/PEG/CCCTCTCATAGGGAGACCUAAG SEQ ID NO: 122CAUCUAAACUA(Y)UUCGACAGGAGGCUCACAACAG GC3′in which Y is one of the sequences provided in FIG. 4 (SEQ ID NOS:86–119), U is 5-(4-pyridylmethyl) uridine, the underlined sequence isDNA, and the 5′ end of the DNA is coupled to a 2,000 MW PEG linker,which in turn is coupled to the free amine of diene reactant 17 havingthe structure:

Each of the aforementioned RNA biocatalysts (hereinafter referred tocollectively as “8I biocatalysts”) may be used to provide a compositionwith anti-PBP2a activity. Specifically, a free reactant library issynthesized by cyclotrimerizing, with all of the B alkynes and all ofthe C alkynes, the monobactam alkyne (A alkyne 24) having the formula:

Cyclotrimerization produces a free reactant sublibrary, termed C24,comprising a large number of monobactams. Following cyclotrimerization,the free reactant sublibrary may be partitioned from unreacted reagents.The free reactant sublibrary C24 is then incubated with one or more ofthe aforementioned 8I RNA biocatalysts (including tethered dienereactant 17). Biocatalysis yields a composition having anti-PBP2aactivity. The individual biocatalyzed products in the composition withanti-PBP2a activity may then be purified by virtue of their affinity forPBP2a. The structure of the product(s) responsible for the anti-PBP2amay be determined by, for example, a combination of reactant librarydeconvolution, involving the synthesis and analysis of successivelysmaller subsets of free reactant sublibrary C43, and tandem C18reversed-phase HPLC-electrospray mass spectrometry (LC-VMS) techniques.

Although the monobactams with anti-PBP2a activity described throughoutthis application were initially identified using RNA biocatalysts, oneskilled in the art will appreciate that it is possible to synthesize allof the aforementioned monobactams using standard organic synthesistechniques. Hence, the invention is not limited to monobactams withanti-PBP2a activity that are formed by the RNA biocatalysts describedherein.

In another embodiment, the invention provides a monobactam compound withthe following formula:

wherein each n is independently 0–4; each X is independently O, S, CH₂or NH; each R is independently lower alkyl optionally substituted withOR, where R₁ is H or lower alkyl; and each Z is independently H;halogen, OH; phenyl, heteroaromatic, or lower alkyl optionallysubstituted with one or more halogen, OH, phenyl or heteroaromaticgroups. This compound may be synthesized according to the method ofExample 13.

The present methods resulted in the preparation of the monobactamcompounds described above. Those compounds have antibacterial activityand thus may be administered to patients (including humans and mammals)in need thereof. For therapeutic or prophylactic treatment, thecompounds of the present invention may be formulated in a pharmaceuticalcomposition, which may include, in addition to an effective amount ofactive ingredient, pharmaceutically acceptable carriers, thickeners,diluents, buffers, preservatives, surface active agents and the like.Pharmaceutical compositions may also include one or more other activeingredients if necessary or desirable.

The pharmaceutical compositions of the present invention may beadministered in a number of ways as will be apparent to one of ordinaryskill. Administration may be done topically, orally, rectally, nasally,vaginally, by inhalation, or parenterally (including subcutaneous,intramuscular, intravenous and intradermal), for example.

Topical formulations may include ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Oral formulationsinclude powders, granules, suspensions or solutions in water ornon-aqueous media, capsules or tablets, for example. Thickeners,flavorings, diluents, emulsifiers, dispersing aids or binders may beused as needed.

Parenteral formulations may include sterile aqueous solutions which mayalso contain buffers, diluents and other suitable additives.

The dose regimen will depend on a number of factors which may readily bedetermined, such as severity and responsiveness of the condition to betreated, but will normally be one or more doses per day, with a courseof treatment lasting from several days to several months, or until acure is effected or a diminution of disease state is achieved. One ofordinary skill may readily determine optimum dosages, dosingmethodologies and repetition rates. In general, it is contemplated thatunit dosage form compositions according to the present invention willcontain from about 0.01 mg to about 500 mg of active ingredient,preferably about 0.1 mg to about 10 mg of active ingredient. Topicalformulations (such as creams, lotions, solutions, etc.) may have aconcentration of active ingredient of from about 0.1% to about 50%,preferably from about 0.1% to about 10%. However, final strength of thefinished dosage form will depend on the factors listed above and may bereadily determined by one of ordinary skill.

While the present invention has been described in terms monobactamderivatives having activity against MRSA, it will be readily appreciatedthat the methods described herein could be used to generate compoundshaving different core structures, as well as compounds active againstother pharmaceutical targets.

EXAMPLE 1

Reactant Library Design and Synthesis

The PBP2a Evolutionary Chemistry™ small molecule reactant library wasdesigned around the monobactam pharmacophore. An approximate5,000,000-member monobactam library was assembled and surveyed for PBP2ainhibitors using the Evolutionary Chemistry process. The monobactamlibrary was created by the RNA catalyzed combination of free andtethered reactants. The tethered reactants attached to the RNA via a PEGlinker were the following 20 dienes (diene reactants 1–20):

Example 6 below describes the manner in which the dienes were tetheredto RNA.

Because no measures were taken in the first series of selections toeliminate reactions between free reactants and functional groupsinherent to the RNA, the following nucleotide units that make up the RNAcould be considered tethered reactants as well.

The free reactants were assembled via the cyclotrimerization of threedifferent classes of alkynes consisting of monobactam alkynes (alkyneA's), dienophiles/reactant alkynes (alkyne B's) and alkynes bearingother functionality (alkyne C's). The alkynes used to create themonobactam free reactant library are shown below.

-   A Alkynes: 30 monobactam (A) alkynes were synthesized and used to    create the free reactant library. The 30 monobactam alkynes were    comprised of all of the combinations of R₁ and R₂ as shown in Table    3:

TABLE 3 A alkynes (monobactam alkynes) R₁ R₂

H

A10 A11 A12 A13 A14

A20 A21 A22 A23 A24

A30 A31 A32 A33 A34

A40 A41 A42 A43 A44

A50 A51 A52 A53 A54

A60 A61 A62 A63 A64

-   B Alkynes: The following 15 alkynes were used in the synthesis of    the free reactants, each of which has reactive functionality that    RNA can catalytically modify using the tethered reactants.

-   C Alkynes: The following 9 alkynes were used to synthesize the free    reactant library.

The free reactant library was comprised of 30 free reactantsub-libraries with each sub-library created by the cyclotrimerization ofone A alkyne with all of the C alkynes and either with or without all ofthe B alkynes. Specifically, A alkynes A10–A44 were cyclotrimerized withall of the B alkynes and all of the C alkynes, while A alkynes A50–A64were cyclotrimerized only with the C alkynes. Each free reactantsublibrary (also referred to asia “core library”) is referred to usingthe A alkyne used to create that sublibrary e.g., “C43” refers to thesublibrary created by the cyclotrimerization of A alkyne A43 with all ofthe B and C alkynes. The following example illustrates thecyclotrimerization of A alkyne A43 with all the B and C alkynes tocreate free reactant sublibrary C43; the same procedure was used for theother core A alkynes.

EXAMPLE 2

Synthesis of Free Reactant Sublibrary C43 by Cyclotrimerization ofAlkynes

Alkyne A43 has the following structure:

Cyclotrimerization was performed using the following cobalt catalyst,hereinafter referred to as “Cp$Co[COD]”:

This alkyne cyclotrimerization catalyst is described in great detail inU.S. Pat. Nos. 5,659,069; 5,760,266; and 6,225,500, each entitled“Method for the Cyclotrimerization of Alkynes in Aqueous Solutions,” andeach incorporated herein by reference in their entirety.

All of the following weighing and reaction set-ups were conducted in aninert atmosphere glove box.

-   Alkyne A43 (monobactam alkyne): In the box, weigh out 228 mg (0.667    mmol) of core into reaction tube.-   B Alkynes (all 15): Weight out each solid, combine in a clean dry    vial and take into the box. Combine liquids in a flask,    freeze-pump-thaw to remove oxygen (3×), and take into the box.    Dissolve the liquids in dry deoxygenated THF to a final volume of    2.0 mL, transfer the solution to the vial containing the solid    alkynes and store the mixture in the glovebox freezer. Label    contents of bottle as Alkyne B Core 43 Main Library Master Mix with    the date prepared. Final stock concentrations should be 33 mM in    each alkyne and 500 mM total alkyne. Use 2.0 mL in    cyclotrimerization mixture for library synthesis (0.667 mmol core    A43).-   C Alkynes (all 9): Weight out each solid and combine in a clean and    dry vial and take into the box. If necessary, Vac-transfer fresh    batches of the liquids and take into the box. Dissolve the solids in    dry deoxygenated THF to a final volume of 2.0 mL, add remaining    liquid alkynes and store in the glovebox freezer. Label contents of    bottle as Alkyne C Core 43 Main Library Master Mix with the date    prepared. Final stock concentrations should be 37 mM in each alkyne    and 333 mM total alkyne. Use 2.0 mL in cyclotrimerization mixture    for main library synthesis (0.667 mmol core A43).-   Cp$Co[COD]: Weigh out 74.4 mg (0.234 mmol) catalyst in glovebox. Add    2.0 mL of dry deoxygenated THF to give a final stock concentration    of 117 mM. Use 2.0 mL in cyclotrimerization mixture for main library    synthesis (0.667 mmol core A43).    Cyclotrimerization Reaction

To Alkyne A in the reaction tube, add 2.0 mL of alkyne B stock solution,2.0 mL of alkyne C stock solution, 2.0 mL of catalyst stock solution and7.3 mL of dry deoxygenated THF to give a final volume of 13.3 mL. Finalconcentrations should be the following:

-   Alkyne A: 50 mM-   Alkyne B (each alkyne): 5.0 mM-   Alkyne C (each alkyne): 5.6 mM-   Cp$Co[COD]: 17.5 mM    Add stir bar, seal reaction tube, take out of box and heat at    105° C. for 72 hr.

The cyclotrimerization reaction products were then subjected to anactivated charcoal treatment according to the following method:

Activated Charcoal Treatment

-   Equipment:    -   100 mL round bottoms    -   THF    -   MeOH    -   Clean rotovap bump trap    -   Transfer pipet    -   10 mL syringe    -   Acrodisc (0.45 μM, PTFE)-   Procedure: Transfer cyclotrimerization reaction to 100 mL round    bottom flasks. Rotovap with a bath temperature of 20° C. The mixture    may bump. After concentrating add up to 100 mL MeOH in 25 mL to the    flask to dissolve the reaction mixture. If products do not    completely dissolve then roto-vap to dryness and proceed to the next    step, deprotection. If products dissolve completely then add 90 mg    of activated charcoal and a stir bar, cap with a plastic cap-plug    and stir for 5 minutes at room temperature. Using the transfer    pipet, transfer the mixture to the 10 mL syringe fitted with the    acrodisc filter. Filter the mixture into a 100 mL round bottom    flask. Rinse the flask with 3 mL and then 1.5 mL of MeOH, filtering    each through the acrodisc and collecting the filtrate into the 100    mL round bottom flask. Concentrate the mixture on the rotovap using    minimal heat (<25° C.) to avoid bumping. The crude reaction mixture    should be a brown oil.

The crude reaction mixture was then deprotected according to thefollowing procedure:

Deprotection

-   Equipment:    -   Box    -   2×14/20 septa    -   18 gauge disposable needle    -   PdL₄    -   0.2 M TEAA in DMF    -   1 mL pipetman    -   stir bar    -   DMF-   Procedure: Place a septum equipped with the 18 g needle on the    flasks containing the crude reaction mixture and take the flask into    the box. To each of the three flasks add 46 mg of PdL₄ (6 mol %)    followed by 33 mL of 0.2 M TEAA/DMF (10 eq.) solution. Add a stir    bar to each flask and seal with a septum. Remove all from the box    and stir for 4 hours at room temperature. Remove the stir bar and    rinse with DMF. Concentrate the mixture on the rotovap using minimal    heat (<25° C. bath temp.). Further evaporate the DMF on a high    vacuum line for at least 15 minutes while stirring or rotating the    flask to increase surface area.

The deprotected products were then subjected to anion exchangepurification according to the following procedure.

Anion Exchange Purification

-   Equipment:    -   Bio-rad low-pressure chromatography system    -   Sephadex A-25 resin    -   15% MeOH/H₂O    -   15% MeOH/500 mM TBK (pH 7) or 15% MeOH/500 mM NaCl    -   1000 mL round bottom flask-   Procedure: Dilute mixture to <5 mM using 15% MeOH/H₂O. Load onto 200    mL of Sephadex A-25 anion exchange resin (HCO₃ ⁻ of Cl⁻    equilibrated) at 1 mL/min. Wash column with 15% MeOH/H₂O at 15    mL/min for 50 min. Elute library with 15% MeOH/500 mM TBK or 15%    MeOH/500 mM NaCl solution at 15 mL/min., collect sample based on UV    absorbance (usually peak elutes after 10–15 min of TBK or NaCl and    requires ˜30 additional minutes to fully elute). Freeze purified    library and lyophilize.

The residude from the anion exchange purification was then subjected toreverse phase purification according to the following procedure.

Reverse Phase Purification

-   Equipment:    -   Prep-C18 reverse phase HPLC column    -   Prep-HPLC-   Procedure: Dissolve residue from anion exchange purification in a    minimum amount of H₂O. Add MeCN up to 10% to obtain complete    solution of residue. Load onto the prep C18 column at 1% MeCN/H₂O,    wash for 10 minutes with 1% MeCN/H₂O and then flash elute library by    rapidly increasing mobile phase to 95% MeCN/H₂O over 2 minutes.    Collect and combine all products that are eluted with the increased    MeCN, remove MeCN by rotary evaporation and remove remaining H₂O by    lyophilization.

EXAMPLE 3

Creation of Free Reactant Libraries by Combination of Individual FreeReactant Sub-Libraries

Each of the 30 free reactant sub-libraries was dissolved in 50% MeOH/H₂Oto a final concentration of 100 mM. They were combined as follows tocreate the 10 free reactant libraries (FR1–10):

-   Library Stocks: All volumes correspond to 100 mM stock solutions of    each of the sub-libraries in 50% MeOH/H₂O. Final concentration of    total monobactam in each library is 80 mm.-   FR 1: C10, C12, C53 (369 μL C10; 369 μL C12; 62.0 μL C53; 200 μL    H₂O)-   FR 2: C11, C44, C52 (369 μL C11; 369 μL C44; 62.0 μL C52; 200 μL    H₂O)-   FR 3: C21, C43, C64 (369 μL C21; 369 μL C43; 62.0 μL C64; 200 μL    H₂O)-   FR 4: C13, C20, C54 (369 μL C13; 369 μL C20; 62.0 μL C54; 200 μL    H₂O)-   FR 5: C14, C23, C50 (369 μL C14; 369 μL C23; 62.0 μL C50; 200 μL    H₂O)-   FR 6: C34, C42, C51 (369 μL C34; 369 μL C42; 62.0 μL C51; 200 μL    H₂O)-   FR 7: C22, C31, C63 (369 μL C22; 369 μL C31; 62.0 μL C63; 200 μL    H₂O)-   FR 8: C24, C30, C62 (369 μL C24; 369 μL C30; 62.0 μL C62; 200 μL H₂O-   FR 9: C32, C41, C60 (369 μL C32; 369 μL C41; 62.0 μL C60; 200 μL    H₂O)-   FR 10: C33, C40, C61 (369 μL C33; 369 μL C40; 62.0 μL C61; 200 μL    H₂O)-   FR 11:    -   36.9 μL C10, C11, C12, C13, C14 C20, C21, C22, C23, C24 C30,        C31, C32, C33, C34 C40, C41, C42, C43, C44    -   +6.2 μL C50, C51, C52, C53, C54 C60, C61, C62, C63, C64    -   +200 μL H₂O

EXAMPLE 4

Cloning, Expression, and Purification of PBP2a

Following previously published cloning and expression methods (Frank etal., Protein Expr. Purif. 6: 671–8), the mecA gene from MRSA strain 27was modified to remove the putative N-terminal trans-membrane region andcloned into the T7 RNA polymerase expression vector pET-11d, which wasthen used to transform Escherichia coli strain BL21 (DE3). The proteinwas isolated in the form of inclusion bodies, requiring extraction,denaturation, and renaturation by methods described in the abovereference. The protein was then purified by cation-exchange on CMSepharose (Sigma) and affinity chromatography on Reactive Blue 4 agarose(Sigma). Typical yields of purified protein were 5 mg/L culture.

EXAMPLE 5

Conjugation of Purified PBP2a to Sepharose Beads

For use in partitioning reactions, PBP2a was conjugated tosulfhydryl-functionalized Sepharose 4B via the heterobifunctionalcross-linking reagent sulfosuccinimidyl6(3-[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SPDP; Pierce),providing a disulfide linkage between the solid support and PBP2a. Stepone of the conjugation procedure was incubation of 10 mg/ml PBP2a with 5mM Sulfo-LC-SPDP in 150 mM NaCl, 0.05% Triton X-100, and 50 mM sodiumphosphate, pH 7.5. Following a one hour incubation at room temperaturewith constant gentle mixing, unreacted Sulfo-LC-SPDP was removed byextensive washing (with 150 mM NaCl, 10 mM EDTA, 0.05% Triton X-100, 50mM sodium phosphate, pH 7.5). Step two of the procedure was thepreparation of sulfhydryl-functionalized Sepharose beads.Pyridyldithio-functionalized Sepharose 4B (Sigma) was added at 125 mg/mlto 50 mM HEPES, pH 7.5, allowed to completely hydrate, then transferredto a mini-chromatography column (Bio-Rad) and washed extensively withthe same buffer. One ml of 200 mM dithithreitol in 50 mM HEPES, pH 7.5was added per 500-μl bead bed volume and the column was capped.Following a 30-min incubation at room temperature with constant mixingon a rotating platform the beads were extensively washed (˜25 ml) with150 mM NaCl, 10 mM EDTA, 0.05% Triton X-100, 50 mM sodium phosphate, pH7.5. The washed beads were transferred to a microcentrifuge tube andexcess buffer was removed. In step 3 of the procedure, 450-μl ofpyridyldithiol-functionalized PBP2a from step 1 was combined with a500-μl bed volume of thiol-functionalized Sepharose 4B prepared instep2. The reaction was incubated at 4° C. for 16 hours then transferredto a mini-chromatography column and washed extensively (˜20 ml) with 150mM NaCl, 10 mM EDTA, 0.05% Triton X-100, and 50 mM sodium phosphate, pH7.5 to remove non-conjugated PBP2a. Sepharose-S-S-PBP2a bead conjugateswere stored at 4° C.

The quantity of active PBP2a per microliter bead bed volume wasdetermined by active site titration with [¹⁴C]benzylpenicillin. A 10-μlbead bed volume was first washed with three 200-μl volumes of assaybuffer (1.2 M NaCl, 20% glycerol, 20 mM HEPES, pH 7.0).[¹⁴C]benzylpenicillin at 200 μg/ml in assay buffer was added to thebeads and the reaction was incubated at room temperature with constantmixing on a rotating platform for 3 hours. Following incubation, thebeads were washed with five 400-μl volumes of 1.2 M NaCl, 50 mM HEPES,pH 7.0. The beads were then suspended in 100 μl of the same buffer andtransferred to scintillation fluid for liquid scintillation. Thequantity of active PBP2a per microliter bed volume was determined byextrapolation from a standard curve prepared from scintillation countsof [¹⁴C]benzylpenicillin at known concentrations and specific activity.

EXAMPLE 6

Design and Synthesis of Biocatalyst RNA Library

The modified RNA library utilized for each Evolutionary Chemistryexperiment was composed of ten different RNA subpopulations (A–J), eachdiffering in a 5′-encoding sequence that permitted the 5′ ligation ofonly one out of ten different sequence-encoded 10 nt ssDNA-PEG₂₀₀₀-Dienereactant species (see FIG. 5). Each RNA in the population had in commona 6-nucleotide high efficiency T7 transcription initiation sequence, a100-nt contiguous random sequence block (100N), and a 3′-definedprimer-annealing sequence.

The dsDNA template for the initial random sequence modified RNA librarywas generated through high-efficiency PCR amplification ofchemically-synthesized ssDNA with the following sequences:

A: GGGAGACAAGAATAAACGCTCAA-(100N)- SEQ ID NO: 1 TTCGACAGGAGGCTCACAACAGGCB: GGGAGATGCTACTACTAACAACA-(100N)- SEQ ID NO: 2 TTCGACAGGAGGCTCACAACAGGCC: GGGAGGAAACATCACAATCCATA-(100N)- SEQ ID NO: 3 TTCGACAGGAGGCTCACAACAGGCD: GGGAGATAATAAATGCCCAGAGA-(100N)- SEQ ID NO: 4 TTCGACAGGAGGCTCACAACAGGCE: GGGAGAAATACAAATAGGCAGGA-(100N)- SEQ ID NO: 5 TTCGACAGGAGGCTCACAACAGGCF: GGGAGAACTTATTATTCACCCGA-(100N)- SEQ ID NO: 6 TTCGACAGGAGGCTCACAACAGGCG: GGGAGACTATTTATCATACGGCA-(100N)- SEQ ID NO: 7 TTCGACAGGAGGCTCACAACAGGCH: GGGAGAATCAAAGTAATCGCTCA-(100N)- SEQ ID NO: 8 TTCGACAGGAGGCTCACAACAGGCI: GGGAGACCTAAGCATCTAAACTA-(100N)- SEQ ID NO: 9 TTCGACAGGAGGCTCACAACAGGCJ: GGGAGAAGGTAGTAGTAGAAGAT-(100N)- SEQ ID NO: 10TTCGACAGGAGGCTCACAACAGGC

The 5′ primers utilized in this amplification (and throughout theselection experiments) included a T7 RNA polymerase promoter sequenceand have the following sequence:

5pA: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 11 GAC AAG AAT AAA CGC TCA A5pB: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 12 GAT GCT ACT ACT AAC AAC A5pC: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 13 GGA AAC ATC ACA ATC CAT A5pD: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 14 GAT AAT AAA TGC CCA GAG A5pE: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 15 GAA ATA CAA ATA GGC AGG A5pF: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 16 GAA CTT ATT ATT CAC CCG A5pG: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 17 GAC TAT TTA TCA TAC GGC A5pH: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 18 GAA TCA AAG TAA TCG CTC A5pI: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 19 GAC CTA AGC ATC TAA ACT A5pJ: TAA TAC GAC TCA CTA TAG GGA SEQ ID NO: 20 GAA GGT AGT AGT AGA AGA T

Following PCR amplification, the cDNA was transcribed from the T7polymerase promoter. Transcription was performed using5-(4-pyridylmethyl)UTP:

instead of UTP in the transcription reaction (Dewey et al., Nucleosides& Nucleotides 15: 1611–7). Due to the 100-nt contiguous random sequence,each RNA sequence is either unique or, as a result of the aboveamplification process, present in the population at a very low copynumber (<10).

Each 10-nt ssDNA was tethered to one of two possible dienes (approx.equimolar mixture of each) via a 2000-MW polyethylene glycol (PEG)linker, resulting in a total of 20 different diene reactants within theRNA library. The individual diene species are illustrated in Example 1above. The 10-nt ssDNAs have the following sequences and tethereddienes:

Tethered Dienes 10-A: AAA CCA CCC C 1, 2 SEQ ID NO: 21 10-B: CCA GGC ACGC 3, 4 SEQ ID NO: 22 10-C: CTC CTC CTT T 5, 6 SEQ ID NO: 23 10-D: GAGGAG GGA G 7, 8 SEQ ID NO: 24 10-E: GTG TTG GGT G 9, 10 SEQ ID NO: 2510-F: CAC GCG ACA C 11, 12 SEQ ID NO: 26 10-G: TTT CGG CGG G 13, 14 SEQID NO: 27 10-H: GGG TGG TAA A 15, 16 SEQ ID NO: 28 10-I: CCC TCT CAT A17, 18 SEQ ID NO: 29 10-J: ATA GCG GCT C 19, 20 SEQ ID NO: 30

The 10-nt ssDNA species were ligated to the transcribed RNA usingbridging oligonucleotides. Each bridging oligonucleotide (listed below)is complementary to the 5′ portion of only one of the RNAsub-populations and complementary to one of the 10-nt ssDNA molecules.For example, bridging oligonucleotide Br-B was used to ligate ssDNA 10-A(with its two possible attached diene species linked to the 5′ end via a2,000 MW PEG linker) to RNA subpopulation B.

Br-A: CTT GTC TCC CGG GGT GGT TT SEQ ID NO: 31 Br-B: AGC ATC TCC CGC GTGCCT GG SEQ ID NO: 32 Br-C: GTT TCC TCC CAA AGG AGG AG SEQ ID NO: 33Br-D: ATT ATC TCC CCT CCC TCC TC SEQ ID NO: 34 Br-E: TAT TTC TCC CCA CCCAAC AC SEQ ID NO: 35 Br-F: AAG TTC TCC CGT GTC GCG TG SEQ ID NO: 36Br-G: ATA GTC TCC CCC CGC CGA AA SEQ ID NO: 37 Br-H: TGA TTC TCC CTT TACCAC CC SEQ ID NO: 38 Br-I: TAG GTC TCC CTA TGA GAG GG SEQ ID NO: 39Br-J: ACC TTC TCC CGA GCC GCT AT SEQ ID NO: 40

The ligation reaction was carried out in a multiplex formation using thefollowing reaction conditions chosen to greatly minimize heterologousligation: 0.5 μM total modified RNA, 6 μM each of ten different bridgeoligonucleotides, 6 μM each of ten different ssDNA₁₀-PEG₂₀₀₀-Reactants,1× ligase buffer (Boehringer Mannheim), 0.4U/μl T4 DNA ligase, and 8%v/v ligase stability buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mMDTT, 60 mM KCl, 50% glycerol). Following a 3 to 4 hr incubation at 37°C., reaction products were separated by denaturing PAGE. Ligated RNAswere visualized by autoradiography or UV light shadowing, excised fromthe gel, and passively eluted from crushed gel slices. The elutionvolume was spun through a 0.45 micron microcentrifuge spin filter thanwas desalted on a Sephadex size exclusion column NAP column; Pharmacia).

As a result of the ligation, each RNA in a particular subpopulation wastethered to one of two possible dienes. Thus RNA subpopulation Acomprised RNA molecules linked to diene 1 or diene 2, subpopulation Bcomprised RNA molecules linked to diene 3 or diene 4, . . . and RNAsubpopulation J comprised RNA molecules linked to diene 19 or diene 20.

EXAMPLE 7

Biocatalysis, Selection, and Amplification Cycles

Each selection experiment was initiated with 2.4 nmole modified RNAlibrary composed of 240 pmoles of each of RNA subpopulations A–J. Asingle cycle of (1) small molecule library assembly via biocatalysis, 2)selection for small molecule affinity to PBP2a, and (3) amplification ofRNA biocatalysts responsible for assembly of selected small moleculeswas performed as summarized in FIG. 1. Specifically, each of freereactant libraries FR1–10 (described in Example XX and each comprised ofapproximately 4,000 monobactam members) was incubated with a mixture ofRNA subpopulations A–J (a total of 10¹⁵ unique sequences) in thepresence of 200 mM NaCl, 50 mM KCl, 5 mM each of MgCl₂ and CaCl₂, 20 μMeach of CuCl₂ and NiCl₂, 10 μM each of CoCl₂, ZnCl₂, MnCl₂, and FeCl₂,and 50 mM HEPES, pH 7.0, thereby yielding ten separate biocatalyzedproduct libraries (P1–10, wherein the numeral indicates the freereactant library that used in the reaction). Each of P1–10 comprises thebiocatalyzed products (monobactams) tethered to the RNA biocatalyst thatcatalyzed its formation. Biocatalysis reaction parameters for the 17completed cycles are provided in Table 4.

TABLE 4 Biocatalyzed product library assembly reaction parameters. RNAReactant Reaction Biocatalyst Library Duration of Cycle # VolumeConcentration Concentration Incubation at 25° 1 1200 μl  2.0 μM 20 mM 16hours 2  400 μl  1.0 μM 20 mM 14 hours 3  400 μl  1.0 μM 20 mM 12 hours4  200 μl  1.0 μM 20 mM  6 hours  5–14  100 μl  1.0 μM 20 mM  4 hours15–17  200 μl 0.25 μM 10 mM  2 hours

Following the small molecule product assembly reaction, free reactantswere removed on G25 Sephadex and the reacted biocatalyzed productlibrary was concentrated and washed on a 30,000 molecular weight cut-offspin filter.

For selection cycles 1–6 only, the recovered RNA was reverse transcribedwith Superscript II RNaseH⁻ reverse transcriptase (Life Technologies) at46° C. for 45 min. prior to partitioning. The primer oligonucleotideutilized for reverse transcription was complementary to the 3′-definedprimer annealing sequence common to all ten encoded modified RNAsubpopulations; reverse transcription reaction conditions were otherwiseas described by the enzyme supplier. The objective of the selectionprotocol was to capture biocatalyst-coupled monobactam inhibitors ofPBP2a using PBP2a conjugated to beads via a disulfide-containing linker.To discard biocatalysis reaction products with affinity for beadcomponents other than PBP2a, the recovered RNAs were first incubatedwith Sepharose 4B-sulfhydral at 30° C. with constant mixing. Following a1 hour incubation in selection buffer (1.2 M NaCl, 20% v/v glycerol, and20 mM HEPES, pH 7.0), the beads were removed and discarded and thesupernatant was transferred to Sepharose 4B-S-S-PBP2a conjugates forincubation with mixing with the parameters listed in Table 5. The beadswere then transferred to a micro-chromatography column and extensivelywashed prior to release of PBP2a and bound biocatalysts-coupled smallmolecules with 200 mM dithiothreitol (DTT).

TABLE 5 Selection of small molecules with affinity for PBP2a: reactionparameters. Reverse Quantity of Transcription: Bead-Conjugated Durationof Before or After Reaction Active PBP2a in Incubation Cycle #Partitioning? Volume Reaction at 30° 1 Before 750 μl 3825 pmole 6 hours2 Before 250 μl 1275 pmole 6 hours 3 Before 250 μl 1275 pmole 4 hours 4Before 120 μl  480 pmole 3 hours 5–6 Before  60 μl  240 pmole 2 hours 7–14 After  60 μl  240 pmole 2 hours 15–17 After  38 μl  160 pmole 1hour

RNA biocatalysts in the DTT eluate were concentrated and washed on a30,000 molecular weight cutt-off spin-filter then, for selection cycles7–17, reverse transcribed with Superscript II RT.

cDNAs recovered from the selection step were amplified with Taq DNApolymerase (0.07 U/μl) in a multiplex PCR reaction that included a3′-primer complimentary to the 3′-defined sequence of RNA subpopulationsA–J, ten different 5′-primers, each complimentary to the unique 5′region of one of RNA subpopulations A–J present in the initial modifiedRNA library (see appendix for oligonucleotide sequences), and thefollowing: 20 mM Tris-HCl, pH 8.5, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2.25 mMMgCl₂, and 0.2 mM each of dATP, dGTP, dCTP, and dTTP. Thermal cycleparameters were an intial “melt” at 94° C. for 2 min followed by cyclingbetween 94° C. for 30 sec, 66° C. for 30 sec, and 72° C. for 1 min. PCRproducts were purified using QiaQuick spin columns (Qiagen inc.) and themanufacturers recommended protocol.

Purified PCR products were transcribed with T7 RNA polymerase in areaction consisting of 0.05–0.1 μM dsDNA template, 1 mM each of ATP,GTP, CTP, and 5-(4-pyridylmethyl)UTP, 20 mM GMP, 0.1 μCi/μl [α-³²P]ATP,12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 4% glycerol, 0.002% TritonX-100, and 50 mM Tris-HCl, pH 8.0. Following a typically 3-hourincubation at 37° C., transcripts were purified using RNeasy spincolumns (Qiagen, Inc.) and the manufacturer's recommended procedure.

RNA subpopulation-specific 10-nucleotide ssDNA-2000 MW PEG-Dienes wereligated to the 5′-termini of RNAs in a multiplex reaction using T4 DNAligase and a set of 10 different bridging oligos, each uniquelycomplimentary to a single 10-nt ssDNA:RNA subpopulation pair. Ligationreactions consisted of 5 μM RNA, 6 μM each bridging oligonucleotide, 6 Meach 10mer-PEG-Diene pair, RNase inhibitor, T4 DNA ligase buffer(Roche), and 0.4 U/μl T4 DNA ligase. After purification of the ligationproducts by denaturing PAGE, the enriched RNA biocatalyst library wassubjected to the next cycle of biocatalysis, selection, andamplification.

EXAMPLE 8

Analysis of RNA Biocatalysts Obtained from the Evolved BiocatalyzedProduct Libraries

Dideoxynucleotide termination sequencing of post-selection cycle PCRproducts was performed to analyze the evolution of the RNA biocatalyst(modified RNA) libraries over the course of the selection experiments.Reaction products were analyzed by denaturing PAGE; a shift from randomsequence within the 100-nt contiguous random sequence block of theinitial RNA libraries to significant non-randomness within this sequenceblock in evolved RNA libraries was an indication that the RNApopulations were converging on functional sequences (data not shown).

As described above, each of the ten evolved biocatalyzed productlibraries (P1–10) contained ten RNA subpopulations (designated A throughJ as described above) that corresponded, by virtue of their 5′-encodingsequence, to a different pair of diene reactants that were presentduring the selection experiments. The RNA libraries added to the firstselection cycle were composed of an equal molar mixture of these tensubpopulations; the ratio of the ten subpopulations in the evolvedlibraries was expected to provide an indication of the favored dienereactants. Following selection cycle #17, the representation of the tendifferent RNA subpopulations (designated A through J) within each RNAlibrary was determined by the following quantitative PCR procedure: foreach biocatalyzed product library (P1 through P10), the RNA biocatalystswere purified and reverse transcribed, as described above, to yield tencorresponding RNA biocatalyst libraries (B1–B10). Then, a PCR reaction“master mix” containing all components except 5′ primer was prepared andaliquoted to ten reaction tubes. Each of the ten reaction tubes receiveda different radiolabeled 5′-encoded primer (subpopulation specificprimer) and PCR amplification was performed under efficient reactionconditions (linear amplification). Reaction products were separated ondenaturing polyacrylamide gels and analyzed with a PackardInstantImager™. The subpopulation representation data (see Table 6)permitted a focused search for anti-PBP2a biocatalysis products withinthe RNA biocatalyst libraries.

TABLE 6 RNA biocatalyst subpopulation representation. The ratio of eachRNA biocatalyst subpopulation (A–J) within each RNA biocatalyst library(B1–B10) is expressed as a percentage of the total. RNA Biocatalyst RNABiocatalyst Subpopulation Library A B C D E F G H I J B1 51.6 0.01 1.225.03 0.69 0.34 0.26 4.47 36.3 0.0 B2 29.7 0.0 6.30 30.2 2.59 0.32 0.204.96 25.6 0.17 B3 49.3 0.10 8.67 16.7 1.69 0.34 0.17 8.65 14.4 0.09 B438.0 0.00 10.5 22.6 13.6 0.14 0.12 2.13 12.9 0.00 B5 28.3 0.15 1.43 42.53.00 0.28 0.30 3.10 20.8 0.13 B6 46.4 0.11 3.63 27.4 2.62 0.33 0.25 2.9916.1 0.21 B7 66.1 0.19 0.24 8.84 0.21 0.29 0.20 1.05 22.9 0.04 B8 82.00.09 0.04 1.18 0.16 0.17 0.20 1.59 14.5 0.04 B9 77.8 0.29 0.72 2.72 0.771.09 0.80 2.38 13.2 0.20 B10 74.9 0.42 0.62 1.64 0.23 0.18 0.19 1.8020.0 0.03

Alternatively, the evolved RNA biocatalyst libraries B1–10 could beanalyzed according to the following procedure:

-   -   Step 1. An oligonucletide complimentary to the conserved 3′-end        of the RNAs is conjugated to a solid support (microtiter plate        wells, beads, filters, etc). This oligonucleotide, referred to        as the “capture oligo”, will hydridize to all members of the RNA        Library. Following conjugation, the solid support is thoroughly        washed to remove free oligonucleotides.    -   Step 2: “Pre-hybridize” (equilibrate) the solid support with        4×SSC/0.5% sarkosyl buffer for one hour at 60° C.    -   Step 3: Remove pre-hybridization solution and add RNA library        sample in 4×SSC/0.5% sarkosyl buffer. The quantity of added RNA        should exceed (about 1.5×) the capacity of the        conjugated oligonucleotides to ensure saturation. Add        biotin-conjugated oligonucleotide (=5pDETECT oligonucleotide)        that is complimentary to one of the possible 5′-encoded        sequences (a separate hybridization reaction is set up for each        5pDETECT oligonucleotide, each being specific for one of the        possible 5′-encoded sequences).    -   Step 4: Incubate the hybridization reaction for 1 hour at 60° C.    -   Step 5: Remove the hybridization solution and thoroughly wash        the solid support with 1×SSC buffer.    -   Step. 6: To the solid support, add an excess of        streptavidin-alkaline phosphatase conjugate in 1×SSC buffer and        incubate for 30 min at room temperature.    -   Step 7: Remove the streptavidin-alkaline phosphatase conjugate        solution and thoroughly wash the solid support with 1×SSC        buffer.    -   Step 8: To the solid support, add either a chemiluminescent        or\chromogenic alkaline phosphatase substrate. Mix and incubate        at room temperature for a pre-determined period of time (usually        10–30 minutes).    -   Step 9: Record emitted fluorescence with a luminometer (for        chemiluminescent substrates), or absorbance with a        spectrophotometer (for chromogenic substrates), and by        comparison to appropriate controls, calculate the ratio of the        possible 5′-encoded sequences in the RNA library population.

This assay may also be performed with a fluorescently labeled probe, aswould be apparent to one of ordinary skill.

EXAMPLE 9

PBP2a Inhibition Activity of Products from Biocatalyst Subpopulations

Individual evolved RNA biocatalyst subpopulations (A–J) from each ofbiocatalyst libraries B1–10 chosen for further analysis were isolated byPCR amplification of selection cycle 17 PCR products using theappropriate subpopulation-specific 5′-primer. Resulting PCR productswere transcribed with T7 RNA polymerase, transcripts were ligated to theappropriate 10mer-PEG₂₀₀₀-Dienes with T4 DNA ligase, and ligated RNAswere purified by denaturing PAGE. Biocatalysis reactions were performedas described above (see selection cycle methods), except the RNAbiocatalyst subpopulations (0.5 μM) were separately incubated with thethree free reactant sublibraries (10 mM) that comprised the freereactant library utilized during the selection cycles (see Examples 1–3for the individual free reactant sublibraries (C10–C44 and C50–C64)present in each free reactant library FR1–10). Following a two hourincubation at 25° C., free reactants were removed as described in theselection procedure and the biocatalysis reaction products weresuspended in 12 μl assay buffer (500 mM NaCl, 0.05% Triton X-100, 20 mMHEPES, pH 7.0). PBP2a was added in 2 μl of assay buffer, bringing thePBP2a and biocatalysts concentrations to 1.1 μM and approximately 2.8μM, respectively. Following a 90 minute incubation at 30° C., 2 μl of0.5 μg/μl [¹⁴C]benzylpenicillin was added to the reaction. Immediatelyfollowing an additional 30 minute incubation at 30° C., the reaction wasterminated by the addition of 200 μl CM Sepharose (50% suspension) in 10mM sodium phosphate, 0.05% Triton X-100, pH 6.0. After a 15 minute roomtemperature incubation with constant mixing, the CM Sepharose beads withbound PBP2a were transferred to a Micro Bio-Spin column (Bio-Rad) andextensively washed to remove unbound benzylpenicillin. The washed beadswere then carefully transferred to scintillation fluid and subjected toscintillation counting. All competition assay experiments included “noRNA” and “no reactant library” controls. Significant reactantsublibrary-specific inhibition was observed with biocatalysis productsfrom the following subpopulations (denoted: [biocatalyst library number][RNA subpopulation]-[diene reactant number])and free reactantsublibraries:

-   -   1. 3H-15 (i.e. RNA biocatalyst library B3, RNA subpopulation H,        diene reactant 15)+free reactant sublibrary C43;    -   2. 3H-16+free reactant sublibrary C43;    -   3. 8I-17+free reactant sublibrary C24; and    -   4. 7I-18+free reactant sublibrary C63

FIG. 6 illustrates the PBP2a inhibition activity of thesesubpopulations. Biocatalysis reactions that included these RNAsubpopulations and other free reactant sublibraries (i.e., the other twosublibrary members of their respective free reactant library) did notyield active inhibitors; therefore, two-thirds of each of these freereactant libraries was omitted as a possible source of thereaction-contributing monobactam substrate. The RNA 3H subpopulation,with free reactant sublibrary C43 demonstrated the highest level ofPBP2a inhibition and was the focus of additional studies.

EXAMPLE 10

Demonstration that RNA Structure is not Involved in the Observed PBP2aInhibition

To demonstrate that RNA biocatalyst subpopulation 3H biocatalysts wereselected for PBP2a inhibition on the basis of their reaction productsand not by an interaction between the RNA and PBP2a, ribonuclease I(RNase I)-digested and undigested biocatalysis reaction products wereassayed for PBP2a inhibiton. The RNase I digestion conditions were shownto reduce the oligoribonucleotide component of the biocatalysts tomononucleotides, and the digestion reaction components themselves wereshown not to inhibit PBP2a (data not shown). The results indicate thatintact RNA is not a component of the inhibition mechanism (FIG. 7).

EXAMPLE 11

Isolation and Analysis of Individual Biocatalysts

Individual biocatalysts present in RNA biocatalyst subpopulation 3H andRNA biocatalyst subpopulation 8I were isolated by cloning into thevector PCR-Script™ Amp using a cloning kit from Stratagene® and themanufacturer's recommended procedure. Vector inserts were sequencedusing a BigDye™ terminator cycle sequencing kit (Applied Biosystems) andreactions were processed by the sequencing facility of National Jewish.Center (Denver, Colo.). Sequence alignments for clones from biocatalystsubpopulations are provided in FIGS. 2 and 4. Sequence alignments wereproduced with Vector NTI® software (Informax, Inc.).

A preliminary phylogenetic comparative analysis was performed on clonesfrom the RNA biocatalyst subpopulation 3H. While an alignment of thesesequences (FIG. 2) reveals a family with very high similarity(suggesting that they were clonally-derived), sequence similaritiesamong the remaining clones are fairly limited. The primary sequencediversity is highlighted by the phylogenetic tree in FIG. 8.

Approximately one-half of the illustrated clones have been screened fortheir ability to catalyze the synthesis of PBP2a inhibitors utilizingmonobactam reactants present in free reactant sublibrary C43;significant PBP2a inhibition was observed with each. FIG. 3 illustratesthe Inhibition of PBP2a by biocatalysis reaction products from RNAsfound in distant branches of the subpopulation 3H phylogenetic tree. Thefinding that diverse sequences generate PBP2a inhibition is suggestiveof the presence of multiple inhibitor structures within this population.

EXAMPLE 12

PBP2a Inhibition Levels

PBP2a inhibition assays described herein permitted a single-pointdetermination of inhibitor K_(i) with assumptions that yield worst-casevalues. In arriving at the inhibition constant, it was assumed that 100%of the biocatalysts in the biocatalysis reaction had generated product(unlikely), that the full 90 min incubation period was required toachieve the observed level of inhibition (unlikely, but not yetinvestigated), and that the deacylation or off rate (k₃) is 10-foldslower than the acylation or on rate (k₂). IC₅₀ value estimates werederived from the K_(i) values and knowledge of approximate molecularweight.

EXAMPLE 13

Synthesis of Compound (j)

Compound (j) of the present invention may be prepared following thescheme and reactions conditions outlined below. All chemicals wereobtained from either Aldrich Chemical Co. and used without furtherpurification unless otherwise noted. BF₃.OEt₂ was purchased from Aldrichas the redistilled reagent. All anhydrous reactions were performed underArgon. THF and pyridine were freshly distilled.

The compounds in this scheme may be synthesized as follows:

(2). To a mixture of (R)-phenylglycine (10.0 g, 66.0 mmoles) and allylalcohol (17.95 mL, 264 mmoles) in benzene (150 mL) was added p-toluenesulfonic acid (16.4 g, 86.0 mmoles). The reaction round bottom flask wasfitted with a Dean-Stark apparatus and reflux condenser and the mixturewas heated to (105° C.) for 18 hr. The mixture was then cooled uponwhich it became a white solid and the remaining solvent removed byrotary evaporation. EtOAc (500 mL) was added and the solution, in a 1 LErlenmeyer, was cooled in an ice bath. While stirring, saturated Na₂CO₃(300 mL) was added to the solution. The biphasic solution wastransferred to a 1 L separatory funnel, the aqueous layer removed andthe organic layer washed again with saturated Na₂CO₃ (300 mL), brine,and then dried over MgSO₄. The concentrated light brown oil wasdissolved in diethyl ether (300 mL), cooled with an ice bath and whilestirring, 2.0 M HCl (obtained from Aldrich Chemical Co and stored in therefrigerator) in ether was slowly added. The salt product precipitatedout and excess 2.0 M HC 1 in ether was added. The precipitate wascollected, washed with cold diethyl ether, and dried in a dessicatorover P₂O₅ under high vacuum overnight yielding 13.88 g (92% yield) ofsolid product.

(3). A mixture of glycinate ester 2 (13.88 g, 60.96 mmoles),formaldehyde (5.95 mL of 37 wt. % solution in water, 73.15 mmoles) andEt₃N (10.2 mL, 76.15 mmoles) in THF (200 mL) were stirred for 5 hr atroom temperature followed by vacuum filtration to remove Et₃N⁺HCl⁻.⁴ TheTHF solvent was removed by rotary evaporation and the residue wasdissolved in EtOAc (200 mL), washed with water (2×150 mL), brine, andthen dried over MgSO₄. Upon concentration, triazine 3 was obtained(12.39 g, 99% yield). The compound was further dried in refluxingbenzene, by passing the condensate over activated 3Å molecular sieves(500 mL) for 18 hr.²

(5). To a vigorously stirring solution of N-phthaloylglycine 4 (20.7 g,100.8 mmoles) and oxalyl chloride (13.2 mL, 151.2 mmoles) in anhydrousCH₂Cl₂ (110 mL) were added anhydrous DMF (0.33 mL) dropwise at roomtemperature. The reaction stirred until the evolution of gas had ceased(2 h). The reaction mixture was concentrated and coevaporated withanhydrous CH₂Cl₂ (3×100 mL) to remove residual oxalyl chloride, to give22.5 g (100% crude yield) of phthalimidoacetyl chloride 5 as a lightyellow solid.

(6). To a solution of triazine 3 (12.29 g, 20.16 mmoles) in anhydrousCH₂Cl₂ (80 mL) was added BF₃.OEt₂ (7.66 mL, 60.48 mmoles) at roomtemperature and the reaction stirred for 20 minutes followed by coolingto −50° C. to give a borane-imine complex. In a separate container, asolution of phthalimidoacetyl chloride 5 (100.8 mmoles) in anhydrousCH₂Cl₂ (100 mL) was cooled to −78° C. and pyridine (8.15 mL, 100.8mmoles) added, followed by stirring for 5 minutes (it is imperative thatthe triazine is dry or the imine will not form and the product will bethe N-phthalimidoacetyl amide. The drying apparatus used incorporates acondenser above an addition funnel, containing 3A molecular sieves, thatis connected to a 500 mL round bottom flask equipped with a stir bar).The borane-imine complex was then added and the reaction stirred at −78°C. for 30 minutes, allowed to warm to room temperature and stirred foran additional 2 hr. The mixture was washed with 10% CuSO₄ (2×500 mL),saturated Na₂CO₃ (500 mL), water (500 mL), brine and dried over MgSO₄.The concentrated light yellow residue was dissolved in CH₂Cl₂ (300 mL)and placed in a refrigerator overnight. The N-phthaloylglycineprecipitate was removed by vacuum filtration and desired productisolated by silica gel flash chromatography (35% EtOAc/Hexanes). Typicalyields range from 50–70%.

(7). A solution of β-lactam 6 (1.22 g, 3.125 mmoles) in anhydrous CH₂Cl₂was cooled to 0° C. then methyl hydrazine slowly added. The reactionmixture was allowed to warm to room temperature and stirred for 48 hr.The CH₂Cl₂ solvent was removed by rotary evaporation and the desiredproduct isolated by silica gel flash chromatography (3% MeOH/CH₂Cl₂).The product was obtained with 72% yield.

(8). To a solution of β-lactam 7 (1.64 g, 6.32 mmoles) in anhydrousCH₂Cl₂ was added prop-2-ynyl-carbamic acid 4-nitro-phenyl ester (1.53 g,6.96 mmoles) and N,N-diisopropylethylamine (1.33 ml, 7.59 mmoles). Thereaction mixture was stirred at room temperature for 1 hr. The CH₂Cl₂solvent was removed by rotary evaporation and the yellow residue wasdissolved in EtOAc (200 mL), washed with sodium carbonate until organiclayer becomes colorless then dried over MgSO₄. The CH₂Cl₂ solvent wasremoved by rotary evaporation and the desired product isolated by silicagel flash chromatography (ramped from 30% EtOAc/Hexanes to 55%EtOAc/Hexanes). The product 8 was obtained with 56% yield.

(10). In an inert atmosphere glovebox, monobactam alkyne 8 (0.667 mmol),one or more other alkyne (total amount 1.33 mmol) and the cobaltcyclotrimerization catalyst 9 were combined in a sealable reactionvessel with 13.3 mL of dry deoxygenated THF box. The reaction tube wassealed, taken out of the box and heated to 105° C. for 72 hours. Thereaction mixture was then allowed to come to room temperature and theTHF removed under reduced pressure. Approximately 10 mL of MeOH and 75mg of activated charcoal were added and the mixture was stirred atambient temperature for 5 minutes. The mixture was filtered ant thefiltrated concentrated under reduced pressure to give a reddish brownresidue. The products were carried on to the next step without furtherpurification.

(11). In an inert atmosphere glovebox, the cyclotrimerization products10 were dissolved in 0.2 M TEAA in DMF (30.75 ml, 6.15 mmoles).Tetrakistriphenylphosphine palladium (46 mg, 0.07 equiv.) was added tothe reaction and the reaction was left to stir 15 hr outside theglovebox at ambient temperature. The DMF solvent was removed by highvacuum rotary evaporation and the products were purified byanion-exchange chromatography and HPLC.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein: wherein X isCH₂, NH, or O; R₁, is selected from the group consisting of C₁–C₂₀alkyl, C₂–C₂₀ akenyl, C₂–C₂₀ alkynyl, OR⁶, C(O)R⁶, carboalkoxyalkyl, aheterocyclyl selected from the group consisting of furanyl, pyridyl,pyrrolyl and imidazolyl, aromatic hydrocarbon and cycloalkyl, all ofwhich may be optionally substituted by one or more of the groupsselected from C₁–C₂₀ alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl,a heterocyclyl selected from the group consisting of furanyl, pyridyl,pyrrolyl and imidazolyl, aryl, halogen, cyano, nitro, amino, alkylamino,dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,alkylaminoaryl, alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁶, OR⁶,CONR^(6,) wherein all said substituents may be optionally substitutedwith one or more selected from the group consisting of halogen, C₁–C₂₀alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl, OR⁶, C(O)R⁶,carboalkoxyalkyl, cyano, and nitro; R₂, R₃, R₄ and R₅ are independentlyselected from the group consisting of hydrogen, C₁–C₂₀ alkyl, C₂–C₂₀akenyl, C₂–C₂₀ alkynyl, OR⁶, C(O)R⁶, carboalkoxyalkyl, a heterocyclylselected from the group consisting of furanyl, pyridyl, pyrrolyl andimidazolyl, aromatic hydrocarbon and cycloalkyl, all of which may beoptionally substituted by one or more of the groups selected from C₁–C₂₀alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl, a heterocyclylselected from the group consisting of furanyl, pyridyl, pyrrolyl andimidazolyl, aryl, halogen, cyano, nitro, amino, alkylamino,dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,alkylaminoaryl, alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁶, OR⁶,CONR^(6,) wherein all said substituents may be optionally substitutedwith one or more selected from the group consisting of halogen, C₁–C₂₀alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl, OR⁶, C(O)R⁶,carboalkoxyalkyl, cyano, and nitro; and R⁶ is selected from the groupconsisting of hydrogen, halogen, C¹–C²⁰ alkyl, aromatic hydrocarbon, andalkylaryl, wherein all said substituents may be optionally substitutedby one or more carboalkoxy, amino, hydroxyl, carboxyl, lower alkyl,lower alkenyl, lower alkynyl, halo, cyano, nitro, carboxyalkyl, andunsubstituted carbamoyl.
 2. A monobactam with anti-PBP2a activity,wherein said monobactam is prepared by the process comprising: a)providing a monobactam core alkyne having the structure

wherein R₁ is selected from the group consisting of:

R₂ is selected from the group consisting of:

R₃ and R₄ are independently selected from the group consisting ofhydrogen, C₁–C₂₀ alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, OR⁵, C(O)R⁵,carboalkoxyalkyl, heterocyclyl, aromatic hydrocarbon and cycloalkyl, allof which may be optionally substituted by one or more of the groupsselected from C₁–C₂₀ alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl,a heterocyclyl selected from the group consisting of furanyl, pyridyl,pyrrolyl and imidazolyl, aryl, halogen, cyano, nitro, amino, alkylamino,dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl,alkylaminoaryl, alkylcarbonylamino, carboxy, carboxyalkyl, C(O)R⁵, OR⁵,CONR^(5,) wherein all said substituents may be optionally substitutedwith one or more selected from the group consisting of halogen, C₁–C₂₀alkyl, C₂–C₂₀ alkenyl, C₂–C₂₀ alkynyl, cycloalkyl, OR⁵, C(O)R⁵,carboalkoxyalkyl, cyano, and nitro; and R⁵ is selected from the groupconsisting of hydrogen, halogen, C₁–C₂₀ alkyl, aromatic hydrocarbon, andalkylaryl, wherein all said substituents may be optionally substitutedby one or more carboalkoxy, amino, hydroxyl, carboxyl, lower alkyl,lower alkenyl, lower alkynyl, halo, cyano, nitro, carboxyalkyl, andunsubstituted carbamoyl; b) reacting said monobactam alkyne underconditions that promote cyclotrimerization with at least one alkyneselected from the group consisting of:

with the proviso that when R₁ is selected from the group consisting of:

the cyclotrimerization reaction mixture also includes at least onealkyne selected from the group consisting of:

c) performing a Diels-Alder reaction between the cyclotrimerizationproduct of b) and a diene reactant selected from the group consistingof:

whereby a monobactam with anti-PBP2a activity may be prepared.
 3. Amonobactam having the formula:

or pharmaceutically acceptable salt thereof, wherein R₁ is theDiels-Alder product formed by the reaction of

with the functionality on an alkyne selected from the group consistingof:

and wherein R₂ is the functionality on an alkyne selected from the groupconsisting of:


4. A monobactam compound with the following formula:

wherein each n is independently 0–4; each X is independently O, S, CH₂or NH; each R is independently lower alkyl optionally substituted withOR₁, where R₁ is H or lower alkyl; and each Z is independently H;halogen; OH; phenyl, heteroaromatic, or lower alkyl optionallysubstituted with one or more halogen, OH, phenyl or heteroaromaticgroups.